Entry of cells into mitosis characteristically involves coordinated and simultaneous events, which include, for example, cytoskeletal rearrangements, disassembly of the nuclear envelope and of the nucleoli, and condensation of chromatin into chromosomes. Cell-cycle events are thought to be regulated by a series of interdependent biochemical steps, with the initiation of late events requiring the successful completion of those preceding them. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. For instance, at least two stages in the cell cycle are regulated in response to DNA damage, the G1/S and the G2/M transitions. These transitions serve as checkpoints to which cells delay cell-cycle progress to allow repair of damage before entering either S phase, when damage would be perpetuated, or M phase, when breaks would result in loss of genomic material. Both the G1/S and G2/M checkpoints are known to be under genetic control as there are mutants that abolish arrest or delay which ordinarily occur in wild-type cells in response to DNA damage.
The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropriate time. These regulatory proteins can provide exquisitely sensitive feedback-controlled circuits that can, for example, prevent exit of the cell from S phase when a fraction of a percent of genomic DNA remains unreplicated (Dasso et al. (1990) Cell 61:811-823) and can block advance into anaphase in mitosis until all chromosomes are aligned on the metaphase plate (kieder et al. (1990) J. Cell Biol. 110:81-95). In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases. For example, genetic, biochemical and morphological evidence implicate the cdc2 kinase as the enzyme responsible for triggering mitosis in eukaryotic cells (for reviews, see Hunt (1989) Curr. Opin. Cell Biol. 1:268-274; Lewin (1990) Cell 61:743-752; and Nurse (1990) Nature 344:503-508). The similarities between the checkpoints in mammalian cells and yeast have suggested similar roles for cdc protein kinases across species. In support of this hypothesis, a human cdc2 gene has been found that is able to substitute for the activity of an S. pombe cdc2 gene in both its G1/S and G2/M roles (Lee et al (1987) Nature 327:31). Likewise, the fact that the cdc2 homolog of S. cerevisae (cdc28) can be replaced by the human cdc2 also emphasizes the extent to which the basic cell-cycle machinery has been conserved in evolution.
As mitosis progresses, the cdc2 kinase appears to trigger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes, segregation of sister chromatids in anaphase, and cleavage furrow formation. Many target proteins involved in mitotic entry of the proliferating cell are directly phosphorylated by the cdc2 kinase. For instance, the cdc2 protein kinase acts by phosphorylating a wide variety of mitotic substrates such as nuclear lamins, histones, and microtubule-associated proteins (Moreno et al. (1990) Cell 61:549-551; and Nigg (1991) Semin. Cell Biol. 2:261-270). The cytoskeleton of cultured cells entering mitosis is rearranged dramatically. Caldesmon, an actin-associated protein, has also been shown to be a cdc2 kinase substrate (Yamashiro et al. (1991) Nature 349:169-172), and its phosphorylation may be involved in induction of M-phase-specific dissolution of actin cables. The interphase microtubule network disassembles, and is replaced by a mitosis-specific astral array emanating from centrosomes. This rearrangement has been correlated with the presence of mitosis-specific cdc2 kinase activity in cell extracts (Verde et al (1990) Nature 343:233-238). Changes in nuclear structure during mitotic entry are also correlated with cdc2 kinase activity. Chromatin condensation into chromosomes is accompanied by cdc2 kinase-induced phosphorylation of histone H1 (Langan et al. (1989) Molec. Cell. Biol. 9:3860-3868), nuclear envelope dissolution is accompanied by cdc2-specific phosphorylation of lamin B (Peter et al. (1990) Cell 61:591-602) nucleolar disappearance is coordinated with the cdc2-dependent phosphorylation of nucleolin and NO38.
The activation of cdc2 kinase activity occurs during the M phase and is an intricately regulated process involving the concerted binding of an essential regulatory subunit (i.e., a cyclin) and phosphorylation at multiple, highly conserved positions (for review, see Fleig and Gould (1991) Semin. Cell Biol. 2:195-204). The complexity of this activation process most likely stems from the fact that, as set out above, the initiation of mitosis must be keyed into a number of signal transduction processes whose function is to guard against the inappropriate progression of the cell-cycle. In particular, the cell employs such signaling mechanisms to guarantee that mitosis and cytokinesis do not occur unless cellular growth and genome duplication have occurred in an accurate and timely manner.
The cdc2 kinase is subject to multiple levels of control. One well-characterized mechanism regulating the activity of cdc2 involves the phosphorylation of tyrosine, threonine, and serine residues; the phosphorylation level of which varies during the cell-cycle (Draetta et al. (1988) Nature 336:738-744; Dunphy et al. (1989) Cell 58:181-191; Morla et al. (1989) Cell 58:193-203; Gould et al. (1989) Nature 342:39-45; and Solomon et al. (1990) Cell 63:1013-1024). The phosphorylation of cdc2 on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of cdc2 is mediated at least impart by the wee1 and mik1 tyrosine kinases (Russel et al. (1987) Cell 49:559-567; Lundgren et al. (1991) Cell 64:1111-1122; Featherstone et al. (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64:1111-1122).
As the cell is about to reach the end of G2, dephosphorylation of the cdc2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cdc2 complex as a kinase. A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189-196; Lee et al. (1992) Mol Biol Cell 3:73-84; Millar et al. (1991) EMBO J 10:4301-4309; and Russell et al. (1986) Cell 45:145-153). Recent evidence indicates that both the cdc25 phosphatase and the cdc2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al. (1992) Cell 70:139-151; Smythe et al. (1992) Cell 68:787-797; and Solomon et al. (1990) Cell 63:1013-1024. This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of cdc2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of cdc2 and/or a decrease in the rate of its tyrosine phosphorylation. A variety of genetic and biochemical data appear to favor a decrease in cdc2-specific trosine kinase activity near the initiation of mitosis which can serve as a triggering step to tip the balance in favor of cdc2 dephosphorylation (Smythe et al. (1992) Cell 68:787-797; Matsumoto et al. (1991) Cell 66:347-360; Kumagai et al. (1992) Cell 70:139-151; Rowley et al. (1992) Nature 356:353-355; and Enoch et al. (1992) Genes Dev. 6:2035-2046). Moreover, recent data suggest at the activated cdc2 kinase is responsible for phosphorylating and activating cdc25. This event would provide a self-amplifying loop and trigger a rapid increase in the activity of the cdc25 protein, ensuring that the tyrosine dephosphorylation of cdc2 proceeds rapidly to completion (Hoffmann et al. (1993) EMBO J. 12:53).